CN114651175A - Method for determining operating flow rate for a chromatography column in an HPLC system - Google Patents

Abstract

A method for determining an operating flow rate for a chromatography column (4) in an HPLC system (1) is disclosed. The method comprises the following steps: measuring/calculating the pressure of the system (1) without chromatography column (4) for one or more flow rates; fitting a function to the flow rate and the corresponding pressure; a system pressure drop at the predetermined recommended flow rate is calculated from the function and the predetermined recommended flow rate for the chromatography column (4). The operating flow rate is determined by summing the system pressure drop with the maximum column pressure limit and determining the contribution of the system pressure drop to the summed pressure. If the contribution exceeds 1%, the operating flow rate for the column is determined to be the flow rate corresponding to a pressure at the pressure monitor, which is disposed before the column, below a predetermined maximum column pressure limit.

Description

Method for determining the operating flow rate for a chromatographic column in an HPLC system

technical field

This document relates to a method for determining an operational flow rate for a chromatography column in a high performance liquid chromatography system, to a high performance liquid chromatography system comprising a system controller for determining such an operational flow rate, and to a method for carrying out the method Computer programs and computer program products.

Background technique

High performance liquid chromatography (HPLC) systems, such as the ÄKTA™ system from Cytiva™ Life Sciences, are used to separate, identify and quantify compounds, such as biomolecules, from samples consisting of mixtures of compounds. The sample is dissolved in a fluid mobile phase that carries the mixture through an immobile, immiscible stationary phase, typically a column packed with (functionalized) particles, typically 1- 10 microns. Phases are selected based on the affinity of the compound of interest towards the phase. The compounds of the mixture travel through the column at different speeds, causing them to separate. The retention time (the rate of movement of a compound through the medium) varies depending on the strength of the interaction with the stationary phase, the composition of the solvent used and the flow rate of the mobile phase. Compounds separated by the column can be detected by means of mass spectrometry, UV/VIS light absorption, fluorescence, light scattering or refractive index.

The separation capacity of an HPLC system increases with smaller stationary phase particle sizes because the surface area of the phase increases. However, smaller particle sizes increase resistance to flow, making it desirable to use high pressures.

For each column, there is a specific maximum flow pressure that the column can withstand without fracturing. This pressure depends on the material of the stationary phase and the material of the column itself. For each column, there is also a predetermined recommended fluid flow rate at which the separation capacity of the column is optimized.

All components in an HPLC system (ie, flow cells, valves, pumps, columns, etc.) are connected to each other with different tubing. The sample of interest is diluted in the pipeline, which causes a negative impact on the resolution of the system. Resolution will decrease as the pipe diameter increases. Narrow pipes increase resolution, and the disadvantage with narrower pipes is the increased back pressure in the system.

Back pressure is the resistance or force against the desired flow rate of fluid through the system, which results in frictional losses and pressure drop, and thus reduces the fluid flow rate through the column. To compensate for the pressure drop so that the fluid flow rate through the column is maintained at the recommended predetermined flow rate, the pressure applied by the pump may be increased. Thus, there is a risk that the increased pressure will approach or exceed the maximum pressure capability of the HPLC system and the particular column used.

The HPLC system may include: a system pump pressure monitor, which measures the pressure after the system pump; a pressure monitor after the sample pump, which measures the sample pressure; and pressure monitors at the inlet and outlet ports of the column valve, to measure pre-column pressure and post-column pressure. The delta column pressure is the difference between the pre-column pressure and the post-column pressure. A pressure alarm can be set for system pressure after the system pump and/or for incremental column pressure to alert the user that the maximum pressure of the system or column has been reached or that the pressure has exceeded a preset limit. The system can automatically stop when a preset limit has been exceeded.

EP288584 shows a chromatography system comprising only one pressure monitor arranged immediately after the pump. The controller is arranged to estimate the precolumn pressure based on the recorded system pressure, characteristics of the flow path and viscosity and flow rate of the liquid in the system.

It can be difficult for an inexperienced HPLC user to know how to set the operating flow rate to run a chromatography system safely without the risk of an immediate pressure alarm and system shutdown due to the use of too high a flow rate.

SUMMARY OF THE INVENTION

It is an object of the present disclosure to provide a method for determining the operating flow rate for a chromatography column in a high performance liquid chromatography system so that the chromatography system can be operated safely without immediate pressure alarms and system shutdowns due to the use of excessive flow rates risks of. Other objects are to provide a high performance liquid chromatography system including a system controller for determining such operational flow rates, as well as computer programs and computer program products for performing the method.

The invention is defined by the attached independent patent claims. Non-limiting examples arise from the dependent patent claims, the drawings and the following description.

According to a first aspect, there is provided a method for determining an operating flow rate for a chromatography column in a high performance liquid chromatography system. The system includes: a liquid reservoir for the liquid medium; a chromatography column in fluid communication with the liquid reservoir, wherein the chromatography column has a predetermined recommended flow rate and a predetermined maximum column pressure limit; a system pump that can be arranged to force the liquid from the liquid reservoir A fluid flow path, which connects the liquid reservoir, the system pump, and the chromatographic column, at a certain flow rate through the chromatographic column; and a pressure monitor, which is arranged before the chromatographic column. Methods include: a) measuring or calculating the pressure of a system without a column for one or more flow rates; b) fitting a function to the flow rate and corresponding measured or calculated pressure; c) according to the function and for the column and d) by summing the system pressure drop to the predetermined maximum column pressure limit and determining the contribution of the system pressure drop to the summed pressure for the chromatographic column where, if the contribution exceeds 1%, the operating flow rate for the chromatography column is determined to be the flow rate below the predetermined maximum column pressure limit.

The high performance liquid chromatography (HPLC) system may be the ÄKTA™ system from Cytiva™ Life Sciences. The column can be any column known in the art suitable for HPLC systems.

The pressure of the system without the chromatographic column is measured or calculated for one or more flow rates, and a function is fitted to the flow rate and the corresponding measured or calculated pressure. If the flow rate is linear and a flow rate of 0 ml/min corresponds to a pressure of 0 MPa, measuring or calculating only one pressure (ie at the predetermined recommended flow rate for the column) may be sufficient. Otherwise, two or more flow rates may be necessary.

Using the function and the predetermined recommended flow rate for the column, calculate the system pressure drop at the predetermined recommended flow rate.

Back pressure is the resistance or force against the desired flow rate of fluid through the system, which results in frictional losses and pressure drop, and thus reduces the fluid flow rate through the column. To compensate for the pressure drop so that the fluid flow rate through the column is maintained at the recommended predetermined flow rate, the pressure applied by the pump may be increased. Thus, there is a risk that the increased pressure will approach or exceed the maximum pressure capability of the HPLC system and the particular column used.

Determines the operating flow rate for the column in a particular system that compensates for system pressure drop. Determining the operational flow rate may include computation, which may be an iterative process or an approximation. Determining the operating flow rate for the column involves summing the system pressure drop to a predetermined maximum column pressure limit and determining the contribution of the system pressure drop to the summed pressure, where, if the contribution exceeds 1%, the operating flow for the column The rate is determined as the flow rate corresponding to the pressure at the pressure monitor below a predetermined maximum column pressure limit. The pressure at the pressure monitor of the system (with the column) is the combined column pressure and system pressure.

In some embodiments, if the system pressure drop contributes more than 1% to the total pressure, the operating flow rate for the column is determined to be 1-20%, 1-15%, or 1-10% lower than the predetermined maximum column pressure limit The flow rate corresponding to the pressure at the pressure monitor of the system (with the chromatographic column). The operating flow rate for the column is determined to be a flow rate that is sufficiently reduced to avoid transient overpressure alarms in the system. The operating flow rate for the column should also be sufficiently high for the measurement to perform satisfactorily.

If the calculated/measured pressure drop is below 1% or zero, the operating flow rate is maintained at the same flow rate as the predetermined recommended flow rate for the column.

The method described above may be an automatic or at least semi-automatic method. Most features and data can be automatically inserted into the system. Therefore, even inexperienced users can easily perform the method.

With the above approach, HPLC users (and inexperienced HPLC users as well) will be able to set operational flow rates to safely run a chromatography system without immediate pressure alarms (by using excessive flow rates on the system and column) the risk of increased stress in the system).

The pressure of the system can be measured by means of a pressure monitor for one or more flow rates, wherein the chromatography column is replaced or bypassed with tubing.

The system may be provided with valves which may be arranged to automatically bypass the column, thus making the bypass of the column an automatic process. The flow rates used preferably include one or more flow rates at low flow rates (such as 100 μl/min) and one or more flow rates at high flow rates (such as 10 ml/min). The flow rate used depends on which column is to be used in the system.

In step b) of the method, the system in which the chromatography column is disconnected from the system, measured by means of the pressure monitor, may be subtracted from the pressure of the system in which the chromatography column is replaced or bypassed with piping, measured by means of the pressure monitor to calculate the pressure of the system for one or more flow rates.

The system may be provided with valves which may be arranged to automatically bypass the column, thus making the bypass of the column an automatic process. Disconnecting the column from the system means here that the column is not replaced/bypassed with tubing, but rather a gap is provided in the system so that the pressure measured is the pressure of the system before the column. This way of calculating the pressure can be used if the pressure of the system before the column is high (ie, 10% or more higher than the total system pressure without the column (the column is bypassed by piping)). The flow rates used preferably include one or more flow rates at low flow rates (such as 100 μl/min) and one or more flow rates at high flow rates (such as 10 ml/min). The flow rate used depends on which column is to be used in the system.

Alternatively, in step a) of the method described above, the pressure of the system can be calculated for one or more flow rates in the absence of a chromatographic column using Bernoulli's formula, the Bernoulli formula:

ΔP = (128*L*Q*η) / (π*d ⁴ )

where d is the inner diameter (mm) of the fluid flow path, Q is the flow rate (ml/min), L is the length of the flow path (mm), and η is the viscosity of the liquid medium (cP).

In this alternative, the pressure of the system is not measured by a pressure monitor in the system, but is calculated for one or more flow rates for a system without a chromatography column. Fluid flow paths are the tubing used in the system to connect liquid reservoirs, system pumps and chromatography columns, and also other parts of the system (such as flow cells, valves, etc.). Therefore, in order to perform this calculation, some data about the liquid and chromatography system used is required. The flow rates used preferably include one or more flow rates at low flow rates (such as 100 μl/min) and one or more flow rates at high flow rates (such as 10 ml/min). The flow rate used depends on which column is to be used in the system.

The pressure of the system can be measured or calculated without a chromatography column for at least two different flow rates, preferably at least three different flow rates.

A higher number of flow rates can improve the method. The number of different flow rates can be four, five, six or more.

If the determined system pressure drop contributes more than 5%, 10%, 15%, or 20% to the total pressure, the operating flow rate for the column is determined to be the same as the pressure at the system pump below the predetermined maximum column pressure limit corresponding flow rate.

Which level to choose as a lower bound on the contribution of the system pressure drop to the summed pressure (ie, 1%, 5%, 10%, 15%, or 20%) can be determined by theoretical calculations assuming that only from the column The pressure will be very close to the maximum pressure at the predetermined recommended flow rate, and the pressure within the system will have no effect. Defining a lower limit on the contribution of the system pressure drop to the total pressure can avoid unnecessary small drops on the column that would not cause any alarm when operating at a predetermined flow rate. This limit should be set with a safety margin to avoid stalling.

The method may also include calculating/measuring the pressure in the system with respect to the chromatography column by measuring the pressure at the predetermined recommended flow rate with the aid of a pressure monitor and subtracting therefrom the measured or calculated pressure of the system without the chromatography column at the predetermined recommended flow rate. incremental pressure.

Using this method, the incremental pressure can be calculated even if the system is provided with only one pressure monitor arranged before the column. Traditionally, the incremental pressure is calculated by means of a first pressure monitor arranged before the column and a second pressure column arranged after the column. Then, the difference in pressure is the incremental pressure. Adding a pressure monitor to the system after the column can negatively impact the resolution of the chromatography system.

This incremental pressure can be used to protect the column from damage to the stationary phase (packed bed) of the column. The pressure alarm can be set at a predetermined maximum bed pressure, which is typically the pressure for the packed bed (or slightly below that) plus the pressure calculated/measured without the column.

According to a second aspect, there is provided a high performance liquid chromatography system comprising: a) a liquid reservoir for a liquid medium; a chromatography column in fluid communication with the liquid reservoir, the chromatography column having a predetermined recommended flow rate and a predetermined maximum column a pressure limit; at least one system pump that can be arranged to force liquid from the liquid reservoir through the chromatography column at a flow rate; a fluid flow path that connects the liquid reservoir, the system pump, and the chromatography column; and a pressure monitor that is arranged before the chromatography column; and b) a system controller for determining the operating flow rate for the chromatography column of the system, wherein the system controller is arranged to perform steps a) to d) described above.

According to a third aspect, there is provided a computer program comprising program code means for performing the method described above when the program is run on a computer.

According to a fourth aspect, there is provided a computer program product comprising program code means stored on a computer readable medium for performing the method described above when the program is run on a computer.

Description of drawings

Figure 1 schematically shows a high performance liquid chromatography system.

Figure 2 illustrates a method for determining the operating flow rate for a chromatography column in a high performance liquid chromatography system.

Figure 3 shows graphs with measured pressures at different flow rates in a modified ÄKTA™ system without a chromatography column, bypassed using tubing.

Detailed ways

A high performance liquid chromatography (HPLC) system 1 is shown in FIG. 1 . The HPLC system 1 can be used to separate, identify and quantify compounds, such as biomolecules, from samples consisting of mixtures of compounds. The sample 2 can be dissolved/injected in the fluid mobile phase (liquid medium 3 ), which carries the mixture through the chromatography column 4 . The chromatography column can be, for example, a size exclusion column, an affinity column, a hydrophobic interaction column, an ion exchange column or a reversed phase column. The column may have a stationary, immiscible stationary phase, which typically includes packed (functionalized) particles, typically 1-10 μm in diameter.

The compounds of sample 2 traveled through column 4 at different speeds, causing them to separate. The retention time (the rate of movement of a compound through the medium) varies depending on the strength of the interaction with the stationary phase, the composition of the liquid medium used and the flow rate of the mobile phase.

The separation capacity of HPLC System 1 increases with smaller stationary phase particle size because the surface area of the phase increases. However, smaller particle sizes increase resistance to flow, making it desirable to use high pressures.

For each chromatography column 4, there is a predetermined recommended flow rate at which the separation capacity of the column is optimized, which depends on the material of the stationary phase and the material of the column itself. For each column, there is also a maximum column pressure limit, which is the highest pressure the column can withstand without bursting. This pressure depends on the material of the stationary phase and the material of the column itself.

Compounds separated by column 4 can be detected by detector 5 using, for example, mass spectrometry, UV/VIS light absorption, fluorescence, light scattering or refractive index.

The HPLC system 1 also comprises at least one system pump 6 arranged to force liquid from the liquid reservoir 3 through the chromatography column 4 at a certain flow rate.

The pressure monitor 7 measures the pressure after the system pump 6 and before the chromatography column. The exact location of the pressure monitor 7 may vary with different HPLC systems 1 . In some configurations, more than one pressure monitor may be disposed between the system pump 6 and the column 4 .

The fluid flow paths connect the liquid reservoir 3, the system pump 6 and the chromatography column 4 and other components in the HPLC system, ie flow cells, valves, etc. (not shown). The flow path can be composed of different pipes.

The sample is diluted in the pipeline, which causes a negative impact on the resolution of the system. Resolution will decrease as the pipe diameter increases. Narrow pipes increase resolution, and the disadvantage with narrower pipes is the increased back pressure in the system.

Back pressure is the resistance or force against the desired flow rate of fluid through the system 1 , which results in frictional losses and pressure drop, and thus reduces the fluid flow rate through the column 4 . To compensate for the pressure drop so that the fluid flow rate through the column 4 remains at the recommended predetermined flow rate, the pressure applied by the system pump 6 may be increased. Thus, there is a risk that the increased pressure will approach or exceed the maximum pressure capability of the HPLC system and the particular column used.

It can be difficult for an inexperienced HPLC user to know how to set the operating flow rate to run the chromatography system 1 safely without the risk of an immediate pressure alarm and system shutdown due to the use of an excessively high flow rate.

A method 100 for determining an operating flow rate for a chromatography column 4 in the high performance liquid chromatography system 1 of FIG. 1 is shown in FIG. 2 .

The method comprises the following steps: a) Measure 101a or calculate 101b, 101c the pressure of a system without a chromatography column for one or more flow rates.

The pressure of the system 101a can be measured 101a for one or more flow rates without a chromatography column by means of a pressure monitor 7 . If more than one pressure monitor is arranged between the system pump 6 and the column 4, the pressure measured with the pressure monitor 7 arranged closest to the column 4 is the pressure measured 101a in the method. An example of a graph with pressures measured at different flow rates without a chromatography column is shown in FIG. 3 .

No chromatography column 4 here means replacing/bypassing the column with fluid flow paths, piping. The system 1 may be provided with a valve (not shown) which may be arranged to automatically bypass the column 4, thus making the bypass of the column 4 an automatic process.

If the pressure of the system before column 4 is high, the pressure in the system measured by means of the pressure monitor 7 can be obtained by subtracting the pressure of the system in which the chromatography column 4 is replaced or bypassed with piping from the pressure measured by means of the pressure monitor 7 The pressure of the system with the column disconnected from the system to calculate the pressure of the system for one or more flow rates.

Disconnecting the column 4 from the system means here that the column is not replaced/bypassed with piping, but rather a gap is provided in the system so that the pressure measured is the pressure of the system before the column 4.

Alternatively, the pressure of the 101c system can be calculated for one or more flow rates without column 4 using Bernoulli's formula:

ΔP = (128*L*Q*η) / (π*d ⁴ )

where d is the inner diameter (mm) of the fluid flow path, Q is the flow rate (ml/min), L is the length of the flow path (mm), and η is the viscosity of the liquid medium (cP).

In this alternative, the pressure of the system 1 is not measured by the pressure monitor 7 in the system, but is calculated for one or more flow rates for the system without the chromatography column 4 . Fluid flow paths are the tubing used in the system to connect liquid reservoirs, system pumps and chromatography columns, and also other parts of the system (such as flow cells, valves, etc.). Therefore, in order to perform this calculation, some data about the liquid and chromatography system 1 used are required.

In the case where the viscosity is not known, water can be assumed to be used, whereby the viscosity can be estimated for different temperatures using a known expression such as:

V [cP] = A × 10B/(T – C), where T = temperature [K]; A = 0.02414; B = 247.8 K; C= 140 K.

In the next step b) of the method, a function is fitted 102 to the flow rate and the corresponding measured or calculated pressure.

Subsequently, in step c) of the method, the system pressure drop at the predetermined recommended flow rate can be calculated from the function and the predetermined recommended flow rate for the column. The system pressure drop can be calculated from the graph in FIG. 3 as the pressure at a predetermined recommended flow rate, indicated in the graph as A), and the pressure, indicated in the graph as B).

In step d) of the method, the operating flow rate for the chromatography column 4 in the particular system 1 is determined 104, which compensates for the system pressure drop. Determining the operational flow rate may include computation, which may be an iterative process or an approximation. Determining 104 the operating flow rate for the chromatography column 4 includes summing the system pressure drop to a predetermined maximum pressure limit for the column, and determining the contribution of the system pressure drop to the summed pressure. If the contribution exceeds 1%, the operating flow rate for column 4 is determined as the flow rate corresponding to the pressure at the pressure monitor below the predetermined maximum column pressure limit.

In Figure 1, the system controller 8 is arranged for determining the operating flow rate for the chromatography column 4 of the high performance liquid chromatography system 1, wherein the system controller 8 is arranged to perform steps a) to d) discussed above.

With the above method, HPLC users (and inexperienced HPLC users as well) will be able to set operational flow rates to run chromatography system 1 safely without immediate pressure alarms caused by the use of excessive flow rates on the system and column (by through increased stress in the system).

The method may also include measuring the pressure by means of the pressure monitor 7 at the predetermined recommended flow rate and subtracting therefrom the measured or calculated pressure of the system without the chromatography column 4 at the predetermined recommended flow rate using the function from step 102 of the method. to calculate/measure 105 the incremental pressure in system 1 with respect to column 4.

Using this method, the incremental pressure can be calculated even if the system is provided with the only pressure monitor 7 arranged before the column 4 . Traditionally, the incremental pressure is calculated by means of a first pressure monitor arranged before the column and a second pressure column arranged after the column. Then, the difference in pressure is the incremental pressure. The addition of a pressure monitor in system 1 after column 4 can negatively affect the resolution of the chromatography system.

This incremental pressure can be used to protect the column from damage to the stationary phase (packed bed) of the column. The pressure alarm can be set at a predetermined maximum bed pressure, which is typically the pressure used for the packed bed (or slightly below that pressure) plus the pressure calculated/measured without column 4 .

If the incremental pressure is the limiting factor, the one can make the incremental pressure limit alarm increase by the same amount of pressure measured or calculated without the column, rather than reducing the flow rate. This is given that only the components behind the column are included in the calculation/measurement. Alternatively, the flow rate for the entire system without the column can be measured, and the components after the column can then be removed, and thus the pressure generated by these components after the column can be obtained. This increase can only be done as long as the column hardware pressure limit is not exceeded.

Next in Table 1 below is an example of calculations performed using the method described above to determine a series of different columns for use in a retrofitted ÄKTA™ pure system from Cytiva™ Life Sciences4 operating flow rate. The ÄKTA™ pure system was retrofitted by replacing all tubing with tubing with a smaller inner diameter of 0.2 mm, thus resulting in an HPLC system with increased back pressure compared to the ÄKTA™ pure system with the original tubing.

The system pressure is measured 101a or calculated 101b for column 4 at various flow rates using water as the liquid medium. In the graph in Figure 3, the system pressure is shown measured 101a for different flow rates (in the absence of a column). A function is fitted 102 to flow rate and corresponding measured/calculated pressure, FIG. 3 . When using a liquid medium other than water, the person can repeat the test with the specific liquid.

In one example, the predetermined recommended flow rate for column 4 (200 plus 5/150 column from Cytiva™ Life Sciences) is 0.45 ml/min, and column 4 has a maximum column pressure limit of 2 MPa. At the predetermined flow rate indicated as A) in FIG. 3 , the pressure drop indicated as B) in FIG. 3 was 0.32 MPa. The sum of the pressure drop and the maximum column pressure limit is 2.32 MPa. The contribution of the pressure drop to the total pressure is 16%, and thus more than 1%. The operating flow rate for the column was determined as the flow rate corresponding to the pressure at the pressure monitor (ie, the combined column pressure and system pressure) that was 5% below the predetermined maximum column pressure limit (ie, 1.9 Mpa) , and the determined operating flow rate for the column was 0.370 ml/min, which corresponds to a flow rate approximately 18% lower than the predetermined recommended flow rate. The determined operating flow rate may be rounded to a flow rate with no more than two significant figures.

In another example from Table 1, the pressure of the 101b system is calculated as discussed above. The predetermined recommended flow rate for the column (S 75 5/150 column from Cytiva™ Life Sciences) is 0.3 ml/min and the column has a maximum column pressure limit of 1.8 MPa. By subtracting the pressure of system 1 in which the chromatography column is disconnected from the system (used without piping), measured by means of pressure monitor 7, from the pressure of the system in which the chromatography column is replaced or bypassed with piping, measured by means of the pressure monitor to calculate the pressure of the 101b system for different flow rates. A function was fitted to the calculated pressure and corresponding flow rate (not shown, but similar to the graph in Figure 3). The pressure drop at the predetermined flow rate is the difference between the pressure at the pressure monitor 7 (here 0.39 MPa) when arranged to force the liquid through the system 1 without the column (the column is bypassed by piping) and at the predetermined recommended flow rate The difference between the pressure of the system with the disconnected column, here 0.18 MPa, and 0.21 MPa. The sum of the pressure drop and the maximum column pressure limit is 2.01 MPa. The contribution of the pressure drop to the total pressure is 12% and therefore more than 1%. The operating flow rate for the column was determined as the flow rate corresponding to the pressure at the pressure monitor (ie, combined column pressure and system pressure) that was 3% below the predetermined maximum column pressure limit (ie, 1.74 Mpa) , and the determined operating flow rate for the column was 0.26 ml/min, which corresponds to a flow rate approximately 12% lower than the predetermined recommended flow rate.

Table 1

For the calculations shown in Table 1, the contribution limit was set to 10%. Contribution limits can be set as low as 1%. Alternatively, it can be set at a higher limit, such as 15% or 20%. Here, a pressure contribution below 10% is not considered a problem and the determined operating flow rate is kept at the same flow rate as the predetermined recommended flow rate for the column. Which level to choose as a lower limit on the contribution of the system pressure drop to the total pressure can be determined by theoretical calculations assuming that the pressure from the column alone will be very close to the maximum pressure at the predetermined recommended flow rate, while the pressure within the system No effect. Defining a lower limit on the contribution of the system pressure drop to the total pressure can avoid unnecessary small drops on the column that would not cause any alarm when operating at a predetermined flow rate. This limit should preferably be set with a safety margin to avoid system shutdown due to the use of too high a flow rate.

While the above description contains various features, these should not be construed as limiting the scope of the concepts described herein, but rather as merely providing illustrations of some exemplary embodiments of the described concepts. It will be appreciated that the scope of the presently described concepts fully encompass other embodiments that may become apparent to those skilled in the art and that the scope of the presently described concepts should therefore not be limited. References to elements in the singular are not intended to mean "one and only one" (unless expressly stated to be so), but "one or more." All structural and functional equivalents to the elements of the above-described embodiments that are known to those of ordinary skill in the art are expressly incorporated herein and are intended to be encompassed thereby.

Claims (11)

1. A method for determining an operating flow rate for a chromatography column (4) in a high performance liquid chromatography system (1), the system comprising:

a liquid reservoir (3) for a liquid medium,

a chromatography column (4) in fluid communication with the liquid reservoir, the chromatography column having a predetermined recommended flow rate and a predetermined maximum column pressure limit,

a system pump (6), the system pump (6) being arrangeable to force liquid from the liquid reservoir (3) through the chromatography column (4) at a flow rate,

a fluid flow path connecting the liquid reservoir (3), the system pump (6) and the chromatography column (4), and

a pressure monitor (7), the pressure monitor (7) being arranged before the chromatography column (4),

the method comprises the following steps:

a) measuring (101a) or calculating (101b, 101c) the pressure of the system (1) without the chromatography column (4) for one or more flow rates,

b) fitting (102) a function to the flow rates and corresponding measured or calculated pressures,

c) calculating (103) a system pressure drop at the predetermined recommended flow rate from the function and the predetermined recommended flow rate for the chromatography column (4), an

d) Determining (104) an operating flow rate for the chromatography column (4) by summing the system pressure drop with the predetermined maximum column pressure limit and determining a contribution of the system pressure drop to the summed pressure, wherein if the contribution exceeds 1%, the operating flow rate for the chromatography column (4) is determined to be a flow rate corresponding to a pressure at the pressure monitor below the predetermined maximum column pressure limit.

2. The method according to claim 1, wherein the pressure of the system (1) is measured (101a) by means of the pressure monitor (7) for one or more flow rates.

3. The method of claim 2, wherein the chromatography column is replaced or bypassed with tubing.

4. The method of any preceding claim, wherein the pressure of the system (1) is calculated (101b) for one or more flow rates by subtracting the pressure of the system (1) with the chromatography column disconnected from the system, measured by means of the pressure monitor (7), from the pressure of the system, measured by means of the pressure monitor.

5. A method according to any preceding claim, wherein the pressure of the system (1) is calculated (101c) for one or more flow rates without the chromatography column (4) using the bernoulli formula:

ΔP = (128*L*Q*η) / (π*d⁴)

wherein d is an inner diameter (mm) of the fluid flow path, Q is the flow rate (ml/min), L is a length (mm) of the flow path, and η is a viscosity (cP) of the liquid medium.

6. The method of any preceding claim, wherein the pressure of the system (1) is measured (101a) or calculated (101b) without the chromatography column (4) for at least two different flow rates, preferably at least three different flow rates.

7. The method of any preceding claim, wherein if the determined contribution of the system pressure drop to the summed pressure exceeds 5%, 10%, 15% or 20%, the operating flow rate for the chromatography column (4) is determined to be a flow rate corresponding to a pressure at the system pump below the predetermined maximum column pressure limit.

8. The method of any preceding claim, further comprising:

calculating/measuring (105) a delta pressure signal in the system (1) with respect to the chromatography column (4) by measuring the pressure by means of the pressure monitor (7) at the predetermined recommended flow rate and subtracting therefrom the measured or calculated pressure of the system (1) without the chromatography column (4) at the predetermined recommended flow rate.

9. A high performance liquid chromatography system (1), comprising: a) a liquid reservoir (3) for a liquid medium; a chromatography column (4) in fluid communication with the liquid reservoir, the chromatography column (4) having a predetermined recommended flow rate and a predetermined maximum column pressure limit; at least one system pump (6), the at least one system pump (6) being arrangeable to force liquid from the liquid reservoir (3) through the chromatography column (4) at a flow rate; a fluid flow path connecting the liquid reservoir (3), the system pump (6) and the chromatography column (4); and a pressure monitor (7), the pressure monitor (7) being arranged before the chromatography column (4); and b) a system controller (8), the system controller (8) for determining an operating flow rate for a chromatography column (4) of the system (1), wherein the system controller (8) is arranged to perform the steps of any preceding claim.

10. A computer program comprising program code means for performing the method of any one of claims 1 to 8 when said program is run on a computer.

11. A computer program product comprising program code means stored on a computer readable medium for performing the method of any one of claims 1 to 8 when the program is run on a computer.

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